Margaret Arstall

Cytokine-Mediated Apoptosis in Cardiac Myocytes: The Role of Inducible Nitric Oxide Synthase Induction and Peroxynitrite Generation

Increased production of nitric oxide (NO) after induction of the cytokine-inducible isoform of nitric oxide synthase (iNOS or NOS2) in cardiac myocytes and other parenchymal cells within the heart may in addition to contributing to myocyte contractile dysfunction also contribute to the induction of programmed cell death (apoptosis). To investigate the mechanism(s) by which increased NO production leads to apoptosis, we examined the role of NO in primary cultures of neonatal rat ventricular myocytes (NRVMs) after induction by the cytokines interleukin-1beta (IL-1beta) and interferon gamma (IFNgamma) or exposure to the exogenous NO donor S-nitroso-N-acetylcysteine (SNAC) or peroxynitrite (ONOO(-)). Both SNAC (1 mmol/L) and ONOO(-) (100 micromol/L), but not their respective controls (ie, N-acetylcysteine and pH-inactivated ONOO(-)), induced apoptosis in confluent, serum-starved NRVMs at 48 hours. Similarly, incubation of NRVMs with IL-1beta and IFNgamma for 48 hours resulted in an increase in iNOS expression, nitrite production, and programmed cell death. Both the cytokine-induced nitrite accumulation and myocyte apoptosis could be completely prevented by the nonselective NOS inhibitor L-nitroarginine (3 mmol/L) or the specific iNOS inhibitor 2-amino-5, 6-dihydro-6-methyl-4H-1,3-thiazine (AMT, 100 micromol/L). NO-mediated myocyte apoptosis was not attenuated by the inhibition of soluble guanylyl cyclase with ODQ, nor could apoptosis be induced by the incubation of NRVMs with 1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP analogue. However, NO-mediated apoptosis was significantly attenuated by the superoxide dismutase mimetic and ONOO(-) scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP, 100 micromol/L). NO/ONOO(-)-mediated apoptosis was associated with increased expression of Bax with no change in Bcl-2 mRNA abundance. Furthermore, apoptotic cell death was also confirmed in adult rat ventricular myocytes (ARVMs) when grown in heteroculture with IL-1beta- and IFNgamma-treated rat cardiac microvascular endothelial cells. Therefore, cytokine-induced apoptosis in NRVMs and ARVMs is mediated by iNOS induction, ONOO(-), and associated with an increase in Bax levels.

Modulation of the Endothelial Nitric-oxide Synthase-Caveolin Interaction in Cardiac Myocytes IMPLICATIONS FOR THE AUTONOMIC REGULATION OF HEART RATE

The endothelial isoform of nitric oxide synthase (eNOS) is dually acylated and thereby targeted to signal-transducing microdomains termed caveolae. In endothelial cells, eNOS interacts with caveolin-1, which represses eNOS enzyme activity. In cardiac myocytes, eNOS associates with the muscle-specific caveolin-3 isoform, but whether this interaction affects NO production and regulates myocyte function is unknown. We isolated neonatal cardiac myocytes from mutant mice with targeted disruption of the eNOS gene and transfected them with wild-type (WT) eNOS or myristoylation-deficient (myr−) eNOS mutant cDNA. In myocytes expressing WT eNOS, the muscarinic cholinergic agonist carbachol completely abrogated the spontaneous beating rate and induced a 4-fold elevation of the cGMP level. By contrast, in the myr− eNOS myocytes, carbachol failed to exert its negative chronotropic effect and to increase cGMP levels. We then used a reversible permeabilization protocol to load intact neonatal rat myocytes with an oligopeptide corresponding to the caveolin-3 scaffolding domain. This peptide completely and specifically inhibited the carbachol-induced negative chronotropic effect and the accompanying cGMP elevation. Thus, our results suggest that acylated eNOS may couple muscarinic receptor activation to heart rate control and indicate a key role for eNOS/caveolin interactions in the cholinergic modulation of cardiac myocyte function.

Daunorubicin-Induced Apoptosis in Rat Cardiac Myocytes Is Inhibited by Dexrazoxane

-The clinical efficacy of anthracycline antineoplastic agents is limited by a high incidence of severe and usually irreversible cardiac toxicity, the cause of which remains controversial. In primary cultures of neonatal and adult rat ventricular myocytes, we found that daunorubicin, at concentrations </=1 micromol/L, induced myocyte programmed cell death within 24 hours, as defined by several complementary techniques. In contrast, daunorubicin concentrations >/=10 micromol/L induced necrotic cell death within 24 hours, with no changes characteristic of apoptosis. To determine whether reactive oxygen species play a role in daunorubicin-mediated apoptosis, we monitored the generation of hydrogen peroxide with dichlorofluorescein (DCF). However, daunorubicin (1 micromol/L) did not increase DCF fluorescence, nor were the antioxidants N-acetylcysteine or the combination of alpha-tocopherol and ascorbic acid able to prevent apoptosis. In contrast, dexrazoxane (10 micromol/L), known clinically to limit anthracycline cardiac toxicity, prevented daunorubicin-induced myocyte apoptosis, but not necrosis induced by higher anthracycline concentrations (>/=10 micromol/L). The antiapoptotic action of dexrazoxane was mimicked by the superoxide-dismutase mimetic porphyrin manganese(II/III)tetrakis(1-methyl-4-peridyl)porphyrin (50 micromol/L). The recognition that anthracycline-induced cardiac myocyte apoptosis, perhaps mediated by superoxide anion generation, occurs at concentrations well below those that result in myocyte necrosis, may aid in the design of new therapeutic strategies to limit the toxicity of these drugs.

N-acetylcysteine in combination with nitroglycerin and streptokinase for the treatment of evolving acute myocardial infarction. Safety and biochemical effects.

BACKGROUND N-acetylcysteine (NAC) has been shown to potentiate the effects of nitroglycerin (NTG) and to have antioxidant activity. This is the first study to assess the safety and effect of NAC in the treatment of evolving acute myocardial infarction (AMI). METHODS AND RESULTS Patients with AMI received either 15 g NAC infused over 24 hours (n = 20) or no NAC (n = 7), combined with intravenous NTG and streptokinase. Peripheral venous plasma malondialdehyde (MDA), reduced (GSH) and oxidized (GSSG) glutathione concentrations, and rate of reperfusion (using continuous ST-segment analysis) were measured. Cardiac catheterization was performed between days 2 and 5. No significant adverse events occurred. Less oxidative stress occurred in patients treated with NAC than in patients not receiving NAC (GSH to GSSG ratio 44 +/- 25 versus 19 +/- 13 at 4 hours, P < .05). NAC concentration (mean 172 +/- 79 mumol/L at 4 hours) was correlated to GSH concentration (P = .006). MDA concentrations were lower (P = .001) over the first 8 hours of treatment with NAC. There was a trend toward more rapid reperfusion (median 58 minutes, 95% confidence interval [CI] 48 to 98 minutes versus median 95 minutes, 95% CI 59 to 106 minutes; P = .17) and better preservation of left ventricular function (cardiac index 3.4 +/- 0.8 versus 2.6 +/- 0.27 L.min.m2, P = .009) with NAC treatment. CONCLUSIONS NAC in combination with NTG and streptokinase appeared to be safe for the treatment of evolving AMI and was associated with significantly less oxidative stress, a trend toward more rapid reperfusion, and better preservation of left ventricular function.

Inducible nitric oxide synthase and tumor necrosis factor in animal models of myocardial necrosis induced by coronary artery ligation or isoproterenol injection

BACKGROUND Increased expression of inducible nitric oxide synthase (iNOS) has been described in humans with cardiomyopathies. Most animal models of ischemia-induced heart failure use the surgical ligation of coronary arteries. However, studies of iNOS expression in these models may be confounded by a robust immune response because of the surgical procedure itself leading to iNOS expression in the heart, as well as in other tissues. METHODS AND RESULTS iNOS expression was studied in adult male rats injected subcutaneously with either 250 mg/kg of isoproterenol (ISO) or vehicle on 2 consecutive days. This approach induces diffuse myocardial necrosis and leads to the development of a dilated cardiomyopathy. Hearts from ISO-injected animals harvested at 6 weeks had evidence of apical and subendocardial scarring. These hearts showed a 9.6-fold (left ventricle [LV], P = .004) and an 11.9-fold (right ventricle, P = .002) increase in the expression of tumor necrosis factor (TNF), and a 6.8-fold increase (LV, P = .0183) in iNOS messenger RNA compared with vehicle-injected controls. iNOS protein also was detectable by immmunoprecipitation in left ventricular muscle from ISO-injected animals, as well as by immunohistochemical analysis. CONCLUSION Expression of TNF and iNOS in the heart is increased in an experimental model of dilated cardiomyopathy that minimizes the confounding effects of surgery, supporting a role for the activation of innate immunity signaling pathways in the pathogenesis of heart failure.

Early use of N-acetylcysteine with nitrate therapy in patients undergoing primary percutaneous coronary intervention for ST-segment-elevation myocardial infarction reduces myocardial infarct size (the NACIAM trial [N-acetylcysteine in acute myocardial infarction])

Background: Contemporary ST-segment–elevation myocardial infarction management involves primary percutaneous coronary intervention, with ongoing studies focusing on infarct size reduction using ancillary therapies. N-acetylcysteine (NAC) is an antioxidant with reactive oxygen species scavenging properties that also potentiates the effects of nitroglycerin and thus represents a potentially beneficial ancillary therapy in primary percutaneous coronary intervention. The NACIAM trial (N-acetylcysteine in Acute Myocardial Infarction) examined the effects of NAC on infarct size in patients with ST-segment–elevation myocardial infarction undergoing percutaneous coronary intervention. Methods: This randomized, double-blind, placebo-controlled, multicenter study evaluated the effects of intravenous high-dose NAC (29 g over 2 days) with background low-dose nitroglycerin (7.2 mg over 2 days) on early cardiac magnetic resonance imaging–assessed infarct size. Secondary end points included cardiac magnetic resonance–determined myocardial salvage and creatine kinase kinetics. Results: Of 112 randomized patients with ST-segment–elevation myocardial infarction, 75 (37 in NAC group, 38 in placebo group) underwent early cardiac magnetic resonance imaging. Median duration of ischemia pretreatment was 2.4 hours. With background nitroglycerin infusion administered to all patients, those randomized to NAC exhibited an absolute 5.5% reduction in cardiac magnetic resonance–assessed infarct size relative to placebo (median, 11.0%; [interquartile range 4.1, 16.3] versus 16.5%; [interquartile range 10.7, 24.2]; P=0.02). Myocardial salvage was approximately doubled in the NAC group (60%; interquartile range, 37–79) compared with placebo (27%; interquartile range, 14–42; P<0.01) and median creatine kinase areas under the curve were 22 000 and 38 000 IU·h in the NAC and placebo groups, respectively (P=0.08). Conclusions: High-dose intravenous NAC administered with low-dose intravenous nitroglycerin is associated with reduced infarct size in patients with ST-segment–elevation myocardial infarction undergoing percutaneous coronary intervention. A larger study is required to assess the impact of this therapy on clinical cardiac outcomes. Clinical Trial Registration: Australian New Zealand Clinical Trials Registry. URL: http://www.anzctr.org.au/. Unique identifier: 12610000280000.

Randomized Comparison of High-Sensitivity Troponin Reporting in Undifferentiated Chest Pain Assessment

Background—High-sensitivity troponin T (hs-TnT) assays promise greater discrimination of evolving myocardial infarction, but the impact of unguided implementation on the effectiveness of care is uncertain. Methods and Results—We evaluated the impact of hs-TnT reporting on care and outcome among chest pain patients presenting to 5 emergency departments within a multicenter randomized trial. Patients were allocated to hs-TnT reporting (hs-report) or standard reporting (std-report; Roche Elecys). The primary end point was death and new or recurrent acute coronary syndrome by 12 months. A total of 1937 patients without ST-segment elevation were enrolled between July 2011 and March 2013. The median age was 61 (interquartile range, 48–74) years, and 46.3% were women. During the index hospitalization, 1466 patients (75.7%) had maximal troponin <30 ng/L within 24 hours. Randomization to hs-report format did not alter the admission rate (hs-report: 57.7% versus std-report: 58.0%; P=0.069). There was no difference in angiography (hs-report: 11.9% versus std-report: 10.9%; P=0.479). The hs-reporting did not reduce 12-month death or new/recurrent acute coronary syndrome in the overall population (hs-report: 9.7% versus std-report: 7.2% [hazard ratio, 0.83 (0.57–1.22); P=0.362]). However, among those with troponin levels <30 ng/L, a modest reduction in the primary end point was observed (hs-report: 2.6% versus std-report: 4.4%, [hazard ratio, 0.58; 95% confidence interval, 0.34–0.1.00; P=0.050). Conclusions—High-sensitivity troponin reporting alone is associated with only modest changes in practice. Clinical effectiveness in the adoption of high-sensitivity troponin may require close coupling with protocols that guide interpretation and care. Clinical Trial Registration—URL: http://www.ANZCTR.org.au. Unique identifier: ACTRN12611000879965.

Clinical determinants of acetylcholine-induced coronary artery spasm in Australian patients

⁎ Corresponding author at: The Queen Elizabeth Ho University of Adelaide, 28 Woodville Road, Woodville Sou E-mail address: john.beltrame@adelaide.edu.au (J.F. B 1 This author takes responsibility for all aspects of the r of the data presented and their discussed interpretation. 2 This author was involved in the conception and design data, critically revised the article for important intellectual of the version to be submitted.

Incidence of adverse events during treatment with verapamil for suspected acute myocardial infarction

Abstract The potential clinical role of non-dihydropyridine calcium antagonists, such as verapamil and diltiazem, in the management of patients during and subsequent to acute myocardial infarction (AMI) is an area of considerable and changing controversy.1 Recent investigations suggest that both verapamil1,2 and diltiazem1,2,4 exert beneficial effects after AMI, largely, if not entirely mediated by reduction in the incidence of reinfarction. However, there is considerable concern about the use of these negatively inotropic calcium antagonists in patients with significantly impaired left ventricular systolic function.1,2,4 Although diltiazem may be beneficial in the periinfarct period3 and verapamil may exert beneficial effects on both ischemia5 and infarct size6 in this setting, the results of the only large randomized study conducted to date concerning the early use of verapamil — Danish Verapamil Infarction Trial-I (DAVIT-I)1— discouraged initiation of verapamil therapy in the first week, representing the period of maximal risk for reinfarction. Specifically, in DAVIT-I the incidence of death due to cardiogenic shock or pulmonary edema, or both, complicating infarction was 5.3%, somewhat higher than in the placebo-treated patients (3.2%). These results of DAVIT-I may have been influenced by the use of large intravenous doses of the drug and by the absence of thrombolytic therapy, which would be expected to both reduce the potential risk of the development of cardiogenic shock and increase the risk of reinfarction. Therefore, there is a need to reevaluate this area of therapeutics. We prospectively examined the incidence of potential drug-associated adverse events in consecutive patients treated with intravenous or oral verapamil, or both, in the early management of suspected AMI. We also sought to determine potential clinical parameters predisposing to the occurrence of such adverse outcomes. End points included death, significant bradyarrhythmia requiring treatment due to hemodynamic compromise and symptomatic left ventricular failure. Frequency of these outcomes was expressed as a percentage with 95% confidence intervals (CI). Correlates between 12 prospectively chosen clinical variables and individual end points were determined using multiple logistic regression.

Meta-analysis of admission hyperglycaemia in acute myocardial infarction patients treated with primary angioplasty: a cause or a marker of mortality?

AIMS Admission hyperglycaemia (AH) has been associated with worse outcomes in acute myocardial infarction (AMI). In the current review, we evaluated the impact of primary angioplasty (pPCI) on mortality in AMI patients with AH. Our second aim was to evaluate if AH is a marker of baseline risk or an independent predictor of mortality. METHODS AND RESULTS A comprehensive search of four major databases was performed. We included original research studies reporting data on mortality in AMI patients with AH (mean plasma glucose >156 mg/dL/8.7 mmol) and euglycaemia who were treated with pPCI. Of 481 citations, 12 studies were included in the analysis. Admission hyperglycaemia was associated with a higher 30-day [risk ratio (RR) 4.30, P < 0.0001] and 1- to 3-year mortality (RR 2.26, P < 0.0001). As well, AH was more prevalent in women and in patients with an increasing number of cardiac risk factors or angiographic predictors of mortality, such as previous AMI (RR 0.89, P = 0.01), multivessel coronary disease (RR 0.72, P = 0.0001), and involvement of left anterior descending artery (RR 0.92, P < 0.0001). Moreover, patients with AH had larger infarcts (higher creatine kinase-MB; P = 0.004) and more frequent ventricular arrhythmias (P = 0.002). CONCLUSION Despite rapid revascularization and treatment of hyperglycaemia, patients with AH continue to have a higher mortality. Admission hyperglycaemia occurs more commonly in patients who have traditional predictors of worse outcomes-specifically prior infarction, anterior wall infarctions, and multivessel disease. Likely, AH is a predictor of rather than a bona fide therapeutic target in AMI.